CN101112409A - Preparation of phenolic acid valid target in dandelion and use thereof for inhibiting influenza virus - Google Patents
Preparation of phenolic acid valid target in dandelion and use thereof for inhibiting influenza virus Download PDFInfo
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Abstract
The present invention relates to a dandelion plant extract for the prevention and treatment of influenza virus infection diseases and the preparation method, as well as the medical application. The dandelion plant extract of the present invention is prepared by ethanol-water extraction, column chromatography, ethanol solvents elution and refining of the fresh or dry dandelion plant, the content of the phenolic compounds in the extract which are taken as the active ingredients is more than 20 percent. The dandelion plant extract of the present invention has significant anti-influenza virus effect, which can be used for the prevention and treatment of influenza caused by virus infection and related symptoms.
Description
Technical field
The present invention relates to medical technical field, particularly, the present invention relates to a kind of Herba Taraxaci plant extract and preparation method thereof, this extract can expect to have the medicinal usage as the relevant disease that prevents and treat influenza infection to cause.
Technical background
Influenza virus has hyperinfection, thus very easily take place popular, or even worldwide being very popular.1917-1919, outburst influenza in Europe causes 2,000 ten thousand people's death, be a most serious in history flu outbreak, and the death toll of the World War I is 8,500,000 people.China also is the multiple state of influenza, and 3 worldwide being very popular since nineteen fifty-seven are all originated from China.Influenza Virus orthomyxovirus section, Influenza Virus comprises first, second, the third three types, first type antigenic variability is the strongest, infects human and other animals, in causing, the severe disease, attacks all age group crowds, often causes worldwide being very popular.B-mode variability a little less than, only infect humanly, generally cause slight disease, mainly attack the child, can cause local outburst.The third type antigenicity is more stable, only causes infant infection and adult's Sporadic cases.
Because some restrictions that influenza virus vaccine is used, and this vaccine is to characteristics such as clinical influenza patient are invalid, and antiviral treatment plays crucial effect equally in the control of influenza.Antiviral drugs can be divided into ion channel M2 blocker, neuraminidase inhibitor, influenza virus receptor blocking agent and resisiting influenza virus antisense oligonucleotide etc. according to them at the different links of virus replication.What enter clinical practice at present is mainly ion channel M2 blocker and neuraminidase inhibitor.Ion channel M2 blocker mainly contains two kinds: amantadine and rimantadine.They all can produce significant gastrointestinal tract, central nervous system's side reaction, as nervousness, anxiety, absent minded, headache, nausea and vomiting etc.These side effect are generally lighter, how to occur in several hours after medication, can rapidly disappear mostly after the drug withdrawal.Because it is active that ion channel M2 blocker lacks the Type B influenza virus, easily produce drug resistance, and toleration is poor.So they are not widely used.Neuraminidase inhibitor: the neuraminidase inhibitor that enters clinical practice at present has zanamivir and Oseltamivir, but does not find the specific medicament of treatment influenza infection disease up to now as yet.
Chinese traditional medicine is a resourceful treasure-house.Over thousands of year, our ancestors have found much effectively can prevent and treat clinically flu and antiviral natural drug, and these Chinese herbal treatments of life-time service and to influenza and other viral diseases.Though over thousands of year influenza pandemic is arranged within Chinese territory but does not have explosive death record to illustrate that also the preventive effect of Chinese medicine and therapeutical effect are very definite.Under the historical background that severe acute respiratory syndrome (SARS) and bird flu are wreaked havoc now, it is very necessary how using modern disease poison technological development modern Chinese medicine of science novel anti-virus preparation.
Herba Taraxaci is feverfew Herba Taraxaci (Taraxacum mongolicum Hand-Mazz, also claim Mongolian Herba Taraxaci) or belong to the dry herbs of several plants together, be heat-clearing and detoxifying herb, cure mainly acute mastitis, furuncle, conjunctival congestion, pharyngalgia, jaundice due to damp-heat and the puckery pain of pyretic stranguria etc.Herba Taraxaci mainly contains triterpenes, phytosterol, sesquiterpene lactones class, Coumarins, flavonoid, phenolic acid compound, carotenoid and fatty acid.Its pharmacological action has anti-inflammation, antioxidation, hepatic cholagogic, immunomodulating and antitumor etc.At present, injection, tablet and the granule etc. that utilize the Herba Taraxaci single medicinal material to make are widely used in clinical, treat various diseases associated with inflammation and have obtained curative effect preferably.
The pharmacological action of Herba Taraxaci is mainly (1) broad-spectrum antibacterial action, and staphylococcus aureus, staphylococcus epidermidis, micrococcus catarrhalis, Hemolytic streptococcus etc. are had strong killing action, and trimethoprim (TMP) TMP can strengthen its antibacterial action.In addition, Diplococcus pneumoniae, meningococcus, diphtheria corynebacterium, Bacillus proteus, dysentery bacterium, pseudomonas aeruginosa also there is strong inhibitory action.Tubercule bacillus, anthrax bacillus, helicobacter pylori, dermatophytes, herpes simplex virus, leptospira etc. also had certain inhibitory action.(2) antiinflammatory action: clinically diseases associated with inflammation such as mastitis, hepatitis, bronchitis, pharyngolaryngitis, parotitis, cholecystitis, gastritis are all had curative effect preferably.It is reported, Herba Taraxaci ethyl acetate extract and main component luteolin thereof and luteolin-7-O-β-D-glucoside can suppress mice RAW264.7 macrophage nitric oxide synthetase (iNOS) and the proteic expression of COX-2 (COX-2) [the Hu C that lipopolysaccharide (LPS) stimulates, Kitts D D.Luteolin and luteolin-7-O-glucoside from dandelion flowersuppress iNOS and COX-2 in RAW264.7 cells.Molecular and CellularBiochemistry, 2004,265:107.].(3) antioxidation: the Herba Taraxaci extract total flavones has class superoxide dismutase (SOD) effect, can effectively remove ultra-oxygen anion free radical and hydroxy radical.(4) hepatic cholagogic effect: Herba Taraxaci water extract and injection have the hepatic cholagogic effect, can reduce hepatic injury rat glutamate pyruvate transaminase (ALT) level of tetrachloro-methane induction, and can improve the hepatocellular degeneration necrosis.Water extract and the ethanol extract of reporting Herba Taraxaci in addition in addition have antitumor action and immunoregulation effect etc.
Herba Taraxaci has stronger heat-clearing toxin-expelling functions, have higher research and development and be worth, but domestic research to Herba Taraxaci mainly rests on its crude extract, especially lacks the effective substance correlational study; Antiviral and correlational study do not appear in the newspapers.Domestic patent generally all is to use with the compound recipe form; The rare report that from Herba Taraxaci, extracts effective site and suppress at virus to study.
We have carried out suppressing the research of influenza virus to the different extract position of Herba Taraxaci plant with certain technological means.With positive drug ribavirin (virazole) is that the pharmacodynamics test of contrast shows, the phenolic acid effective kind part of Herba Taraxaci extract is at demonstrating good activity in antivirus test and the zoopery therapeutic test in vivo and in vitro of upper respiratory tract infection disease: cause Testis et Pentis Canis passage cell (MDCK) median toxic concentration (TD
50) be 3.26mg/ml, 90% poisoning concentration is 8.69mg/ml, maximal non-toxic concentration is between 100-1000 μ g/ml.Medium effective concentration (the EC of the phenolic acid effective kind part resisiting influenza virus FM-1 of Herba Taraxaci extract (the Mus lung adapted strain of influenza A virus)
50) be 213 μ g/ml, therapeutic index is 15.3.The maximal non-toxic dosage that the phenolic acid effective kind part of Herba Taraxaci extract causes Embryo Gallus domesticus is TC
0=10mg/ embryo, the minimal inhibitory concentration (MIC) that influenza virus FM-1 is infected Embryo Gallus domesticus is 10mg/ml.Animal acute toxicity test shows that the phenolic acid effective kind part toxic and side effects of Herba Taraxaci extract is very low.We have carried out material base research to this effective site (Herba Taraxaci phenolic acid effective kind part), have found wherein to contain the flavonoid component of four luteolin skeletons; More further achievement is: we find in this extract part (phenolic acid effective kind part) first except that containing traditional phenolic acids active substances such as caffeic acid, ferulic acid and chlorogenic acid, the two caffeoyl guinic acid class compound of phenolic acid active substances that also contain three structural similarities, their structure is: 3, and the 5-O-dicaffeoyl quinic acid; 3, the 4-O-dicaffeoyl quinic acid; 4, the 5-O-dicaffeoyl quinic acid.And such caffeoyl guinic acid compounds is in our antiviral screening in the past, be the active component that effectively suppresses influenza virus through checking, therefore according to the fact that increases of antiviral drug effect after these effective ingredient enrichments, can this Herba Taraxaci effective site of inference be expected to develop the new Chinese medicine that becomes novel inhibition viral infection, and with its have no side effect, advantage such as cheap substitutes existing antiviral class Chinese patent medicine.Wherein, two caffeoyl guinic acid compounds of finding in this kind of plant of our reported first are classes by quinic acid (quinic acid) and two caffeic acids (caffeic acid) by the phenolic acids natural component that the esterification condensation forms, and extensively be present in plant kingdom and the number of significant drug activity is arranged.(Li Xiangping, girth is new, Dong Sun Han, Hao Xiaojiang, Xiao Peigen, caffeoyl guinic acid compounds progress, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2006 for Zhao Yu, Zhao Jun) finishes the present invention thus.
Summary of the invention
The purpose of this invention is to provide the extract that is rich in phenolic acid compound in a kind of Herba Taraxaci plant;
Another object of the present invention has provided a kind of method that is rich in the phenolic acid compound extract in the Herba Taraxaci plant for preparing;
A further object of the present invention has provided the purposes that the extract that will be rich in phenolic acid compound in the Herba Taraxaci plant is used to suppress influenza virus;
Another purpose of the present invention has provided a kind of medicine that is rich in the phenolic acid compound extract or pharmaceutical composition that contains in the Herba Taraxaci plant.It is characterized by in this extract part except that containing caffeic acid, ferulic acid and chlorogenic acid, also contain two caffeoyl guinic acid class compound of phenolic acid active substances of three structural similarities, they are: 3, and the 5-O-dicaffeoyl quinic acid; 3, the 4-O-dicaffeoyl quinic acid; 4, the 5-O-dicaffeoyl quinic acid.
Be rich in the phenolic acid compound extract in the Herba Taraxaci plant among the present invention and contain phenoloid content more than 20% by colorimetric method for determining.
Specific embodiments
Plant is in the past discovered and is contained triterpenes, Coumarins, phenolic acids (flavonoid that comprises phenolic hydroxy group), sesquiterpenoids, phytosterol, carotenoid and long-chain fat acid compounds in the Herba Taraxaci plant.The inventor suppresses the result according to different extract parts to influenza virus and finds for activity index: its crude extract has certain extracorporeal antivirus effect activity, and its antiviral activity composition concentrates on the high polarity phenolic acids position of plant extract, by process optimization, the inventor adopts water or water-alcohol solvent extraction to be aided with multiple positive and negative phase chromatography means and obtains this effective site.Through spectroscopic datas such as a peacekeeping two dimensional NMR wave spectrum, mass spectrum, infrared, ultraviolets, we find that first this high polarity phenolic acid effective kind part contains two compounds, and one is the flavonoid component of four luteolin skeletons; A class is exactly traditional phenolic acids active substances such as caffeic acid and chlorogenic acid again, and two caffeoyl guinic acid class compound of phenolic acid of three structural similarities, and they are: 3, and the 5-O-dicaffeoyl quinic acid; 3, the 4-O-dicaffeoyl quinic acid; 4, the 5-O-dicaffeoyl quinic acid.Modern age, pharmacology test studies show that: the flavone compound of this type of luteolin skeleton and single, double coffee acyl quinic acid compounds have multiple important physiologically active; mainly be at immune effect: show and suppress the synthetic and release of leukotriene; suppress hepatitis B virus and HIV (human immunodeficiency virus); suppress histamine release, thereby can be used for antiinflammatory, antiviral and treatment anaphylactic disease.Find that in addition it has potent antioxidation, suppresses lipoxygenase study of anti-atherogenic effect, platelet aggregation inhibitory activity and effect for reducing blood fat isoreactivity.(document: Li Zusheng, Zhu Zhian, the medical science summary, 2004,10 (4), 249-250).Simultaneously, phenolic acid compound content in effective site such as the flavone compound of the luteolin skeleton among the present invention and coffee acyl quinic acid are quite high, and the total content of phenolic acid effective site in extract has surpassed 20%.
The present invention is more ben to be: our reported first is separated from Herba Taraxaci extract effective site and is obtained three dicaffeoyl quinic acid compounds with efficient physiologically active.It is as follows to derive three dicaffeoyl quinic acid compounds structures through integration analysis such as infrared, mass spectrum, ultraviolet and NMR (Nuclear Magnetic Resonance) spectrum.
Particularly, it is as follows to contain the preparation method of phenolic acid effective kind part of the flavone compound of luteolin skeleton and caffeoyl guinic acid compounds among the present invention: by the herb of Herba Taraxaci through pure water system extract, the column chromatography method purification.Specifically comprise the following steps:
A. the mixed liquid of water-strong polar organic solvent (pure water mixed solvent commonly used) extracts through pulverizing or be cut into the medical material of segment, and concentrated extracting solution reclaims organic solvent and obtains concentrated solution;
B. the concentrated solution that step a is obtained is through macroporous adsorbent resin column chromatography, and elder generation is closely colourless to be washed to, and continues with 30～80% ethanol elutions;
C. above-mentioned ethanol elution being evaporated to does not have the alcohol flavor, adopts spray drying method to carry out drying, obtains the thin uniform effective site of color and luster of powder, and this effective site is for mainly containing the mixture of caffeoyl guinic acid compounds.
In the preparation method among the present invention, the alcohol among the step a can be methanol, ethanol, propanol, butanols, ethylene glycol or their mixture, particular methanol and ethanol.The alcohol water mixed solvent can be alcohol and water with the blended solvent of arbitrary proportion, the mixed solvent of preferred alcohol and water, the ethanol water of especially preferred 30-75%.The column chromatography used medium can be silica gel, reverse phase silica gel, macroporous resin, ion exchange resin, aluminium oxide, polydextran gel (Sephadex) class among the step b.Preferred macroporous resin.Eluting solvent can be acetone, alcohol, water or their mixture.The mixed solvent of preferred alcohol and water carries out gradient elution.Wait to spray the density of drying solution after concentrating among the step c in 1.0～1.4, preferred 1.05～1.25.
Medical material among the present invention can be the arbitrary position of home-made Dandelion plant in any one, it promptly can be Herba Taraxaci (claiming Mongolian Herba Taraxaci again) (Taraxacum mongolicumHand-Mazz), alkali ground Herba Taraxaci (claiming magnificent Herba Taraxaci again) (Taraxacum sinicum Kitag), hot river Herba Taraxaci (claiming white edge Herba Taraxaci again) (Taraxacum platypecidum Diels), Herba Taraxaci ohwiani (Taraxacum ohwianum Kitag), anti-luxuriant Herba Taraxaci (Taraxacumgrypodon Dahlst), common Herba Taraxaci medical material on Xingan's Herba Taraxaci Chinese drug markets such as (Taraxacum falcilobum Kitag), can be dry product or bright product, it can be the root of Herba Taraxaci medical material plant, stem, leaf, flower, seed, skin, fruit also can be their mixture.Preferred herb.The more preferably herb part of Herba Taraxaci (claim not only Mongolian Herba Taraxaci) (Taraxacum mongolicumHand-Mazz) and/or alkali ground Herba Taraxaci (but also claiming magnificent Herba Taraxaci) (Taraxacum sinicumKitag).All be called for short Herba Taraxaci in the present invention's the description.
The inventor finds that the Herba Taraxaci liposoluble ingredient effective part extract that the present invention obtains has the function that suppresses the influenza virus growth; Can be used for the treatment of infection disease that influenza virus causes etc.Thereby may develop resisiting influenza virus class pharmaceutical composition.This pharmaceutical composition can be made with the routine techniques in the pharmaceutical field, can make various dosage forms, as tablet, granule, capsule, oral liquid, drop pill, injection, transdermal patch, aerosol etc.These pharmaceutical compositions can be used for the treatment of influenza infection disease purposes.
Further specify the present invention below by embodiment.The following embodiment of mandatory declaration is used to illustrate the present invention rather than limitation of the present invention.Essence according to the present invention all belongs to the scope of protection of present invention to the simple modifications that the present invention carries out.Except as otherwise noted, the percent among the present invention is percetage by weight.
Embodiment 1Phenolic acid compound and preparation of drug combination in the Herba Taraxaci plant
Get the air-dry sample of 10kg Herba Taraxaci medical material, be cut into segment, with 50% ethanol water heating extraction twice, for the first time with 8 times of amounts, for the second time with 6 times of amounts.Extracted 2 hours for the 1st time, extracted 1.5 hours for the 2nd time, concentrate, be splined on the macroporous adsorbent resin of having handled well, more shallow to be washed to color, it is shallow to color with 50% ethanol elution to continue, the eluent reclaim under reduced pressure, spray drying gets the raw material fine powder, and the phenolic acid compound effective site that this is Herba Taraxaci plant extract among the present invention is designated hereinafter simply as the spray powder raw material.With the spray powder raw material: (starch+microcrystalline Cellulose) is inserted in (can be 0～No. 4 capsule as required) in the capsule after granulate (1: 0.5～1: 3, starch wherein: the scope of microcrystalline Cellulose is 1: 0～0: 1) by a certain percentage, promptly.With the caffeic acid is that standard substance contain phenolic acid compound content by this effective site of colorimetric method for determining, and concrete grammar is:
1. instrument and reagent
The ultraviolet-visible spectrophotometer; 100,000/balance; Methanol, the potassium ferricyanide, ferric chloride, hydrochloric acid are analytical pure; Dodecyl sodium sulfate is a chemical pure.Caffeic acid reference substance and Herba Taraxaci extract spray powder raw material are provided by Haizheng Tianhua Medicine Research Co., Ltd., Zhejiang Prov.
2. method and result
2.1 the preparation precision of reference substance solution takes by weighing caffeic acid reference substance 2.50mg, with anhydrous alcohol solution and be settled to 25ml.
2.2 Herba Taraxaci extract spray powder raw material (11.2mg) is got in the preparation of need testing solution, is transferred in the volumetric flask of 50ml, dissolves and is settled to scale, pipettes in 2ml to the 25ml volumetric flask, adds 70% alcoholic solution to 3ml with dehydrated alcohol or distilled water.
2.3 accurate reference substance solution 0.5,1.0,1.5,2.0, the 2.5ml of drawing of the preparation of standard curve, put in the 25ml measuring bottle, add dehydrated alcohol respectively to 3.0ml, each adding distil water 2.0ml again, the accurate respectively again 0.3% sodium dodecyl sulfate solution 2.0ml that adds, 0.6% potassium ferricyanide-0.9% ferric chloride (1: 1) solution 2.0ml, shake up, placed 5 minutes the dark place, add the 0.1mol/L hydrochloric acid solution to 25ml, shake up, placed 20 minutes the dark place, with retinue solution is blank, measures absorbance at the 500nm place.With the absorbance is vertical coordinate, and the reference substance quality is an abscissa, basis of calculation curve.The results are shown in following table, standard curve is y=0.2068x-0.0331, r
2=0.9998.
Different quality standard substance absorbance result
| Reference substance quality (μ g) | Absorbance (A) |
| 1.0 4.0 9.0 16.0 25.0 | 0.3907 0.7822 1.2034 1.6257 2.0372 |
2.4 sample size is measured the accurate spray powder raw material need testing solution of drawing, and is blank with retinue solution, with the 2.3 described absorbances of measuring at the 500nm place.And by standard curve calculation sample total phenol content.The working sample absorbance is 1.3126, calculates content according to standard curve, and the result is 36.27% in the caffeic acid for standard substance content for total phenols in the Herba Taraxaci extract spray powder raw material.2.5 the mensuration of caffeic acid monomer and luteolin 7-O-β-D-glucoside content of monomer in the sample:
Instrument: Waters Alliance type high performance liquid chromatograph, join automatic sampler and PDA diode array detector.Chromatographic condition: chromatographic column is Symmetry
C18 (5 μ m, 4.6 * 150mm); The detection wavelength is 280nm; 30 ℃ of column temperatures; Flow velocity: 0.8mL/min; Mobile phase: methanol/0.1% acetic acid, gradient elution, mobile phase is composed as follows:
| Time (minute) | Methanol | 0.1% acetic acid |
| 0.0 | ?10％ | 90％ |
| 10.0 | ?10％ | 90％ |
| 15.0 | ?30％ | 70％ |
| 40.0 | ?70％ | 30％ |
| 45.0 | ?70％ | 30％ |
| 55.0 | ?100％ | 0％ |
2.5.1 the drafting of standard curve: precision takes by weighing caffeic acid standard substance 1.90mg, puts in the 5mL volumetric flask 50% dissolve with methanol standardize solution.Precision takes by weighing 2mL and puts in the 10mL volumetric flask; Precision takes by weighing luteolin-7-O-β-D-glucoside standard substance 5.30mg, puts in the 10mL volumetric flask 50% dissolve with methanol standardize solution.Precision takes by weighing 2mL and is equipped with in the 10mL volumetric flask of caffeic acid standard solution 2mL; 50% methanol dilution standardize solution.Be configured to the mixed standard solution of caffeic acid and luteolin-7-O-β-D-glucoside, concentration is respectively 0.076mg/mL and 0.106mg/mL, and is standby.Precision takes by weighing spray powder raw material 103.8mg, puts in the 50mL volumetric flask, and 50% methanol constant volume is to scale, and 0.45 μ m filtering with microporous membrane is standby.
Precision takes by weighing hybrid standard product 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, measures peak area according to above-mentioned chromatographic condition, is abscissa with the sample size, and peak area is a vertical coordinate, draws the standard curve of caffeic acid and luteolin 7-O-glucoside respectively.Getting the caffeic acid regression equation is Y=156750.7+6650425x, r=0.9990; Luteolin 7-O-glucoside regression equation is Y=43636.6+1876428x, r=0.9991.
2.5.2 the assay of spray powder raw material: precision takes by weighing this sample solution 5 μ L, according to chromatographic condition sample introduction under the 2.5.1 item, measure peak area, calculate content according to above-mentioned standard curve, the result shows that caffeic acid content is 0.63% in the Herba Taraxaci extract spray powder material sample, and luteolin 7-O-glucoside content is 1.00%.
2.6 the spectral data of two caffeoyl guinic acid class phenolic acid chemical compounds in the Herba Taraxaci extract spray powder material sample
2.6.1 3,5-O-dicaffeoyl quinic acid (Z
1):
Z
1Be pale yellow powder, be soluble in methanol, water.Weak blue-fluorescence is arranged under 254nm, show yellow green with the reaction of 0.6% ferric chloride methanol solution.Fusing point: 170-172 ℃.(temperature is not proofreaied and correct).[α]
D 28:-198.1 ° of (methanol; C 0.32) ultraviolet spectra UVmax (methanol): 220,247,300sh, 330nm.ESI mass spectrum (m/e): 515,352,191,173.Chemical compound Z
1 1H nuclear magnetic resoance spectrum: instrument: INOVA NMR 400MHz; TMS is interior mark; Solvent: deuterated methanol; Chemical compound Z
1 1Provided the characteristic signal δ 7.04 (2H, br s) of two groups of coffee acyl protons among the H-NMR, 6.94 (2H; br d, J=8.4Hz), 6.75 (2H; d, J=8.4Hz) and trans olefinic proton signal δ 6.33,6.24 (each 1H of two groups of AB types; d; J=16.0Hz) and δ 7.59,7.55 (each 1H, d; J=16.0Hz), show chemical compound Z
1Two coffee acyl segments of middle existence.In addition, 5.39 (1H, m), 5.36 (1H is m) with 3.94 (1H, d, J=4.4) three even oxygen hydrogen and 2.29 (1H, d, J=13.2Hz), 2.16 (1H, d, J=13.2Hz) and 2.16 (2H, m) four proton signals can belong to the signal of seven fat proton of quinic acid part according to its chemical shift and peak shape feature.According to chemical compound Z
1 1The contrast of the chemical displacement value of H and document can this chemical compound of preliminary judgement be 3 among the H-NMR, the 5-O-dicaffeoyl quinic acid.
Chemical compound Z
1 13C nuclear magnetic resoance spectrum: instrument: INOVA NMR 100MHz; TMS is interior mark; Solvent: deuterated methanol; Chemical compound Z
1 13C-NMR and DEPT (distortionless polarization transfer enhancing) spectrogram has provided ten undersaturated methine carbons between δ 114-169 (δ 115.1,115.2,115.6 (2 CH), (116.5 2 CH), 123.0,123.1,146.8,147.0), (δ 127.8 for six fragrant quaternary carbons, 127.9,146.8 (2C), 149.6,149.5) and two α, the carbon atom signal of beta-unsaturated carbonyl (δ 168.9,168.3) has further proved chemical compound Z
1In contain two coffee acyl structure fragments.In addition, also have two mesomethylene carbons (δ 36.1,37.8), the methine carbon (δ 72.6,72.1,70.7) of 3 company's oxygen, a quaternary carbon (δ 74.9) and a carboxylic carbonyl signal (δ 177.8), closely similar with the carbon geochemistry displacement of the quinic acid of reporting in the document, and chemical compound Z
1The C-3 and the C-5 of quinic acid structure fragment have the acidylate displacement, thereby can infer chemical compound Z
1Parent nucleus should be quinic acid, and have two coffee acyl substituent groups to be associated in 3 and 5 of quinic acid parent nucleus respectively.And according to chemical compound Z
1 13The contrast of the chemical displacement value of C and document can this chemical compound of preliminary judgement be 3 among the C-NMR, the 5-O-dicaffeoyl quinic acid.As checkings such as HSQC, HMBC, NOESY, finally determine the caffeoyl guinic acid compounds Z in the Herba Taraxaci extract effective site through the two dimensional NMR spectrum
1Structure is 3, the 5-O-dicaffeoyl quinic acid.
2.6.2 3,4-O-dicaffeoyl quinic acid (Z
2):
Z
2Mass spectrum: anion FABMS shows quasi-molecular ion peak m/z 515[M-1]
-Infrared: chemical compound Z
2IR spectrum and chemical compound Z
1Extremely similar, it is shown with hydroxyl (3420cm
-1), ester group (1696cm
-1), phenyl ring (1603,1523cm
-1) and two key (1632cm
-1).
1H,
13C and DEPT spectrum can be released chemical compound Z
2Molecular formula be C
25H
24O
12, chemical compound Z
2 1Provided among the H NMR two groups of coffee acyl protons characteristic signal δ 7.08 (1H, brs), 7.07 (1H; brs), 6.95 (2H, m); 6.78 (2H, d, J=8.4Hz) and the trans olefinic proton signal δ 6.32 of two groups of AB types; 6.30 (each 1H; d is J=16.0Hz) with δ 7.61,7.57 (each 1H; d, J=16.0Hz).
13C NMR and DEPT spectrum have provided ten undersaturated methine carbons between δ 114-169 (δ 115.0,115.2 (3CH), (116.5 2 CH), 123.2 (2 CH), 147.3 (2CH), (δ 127.7 for six fragrant quaternary carbons, 127.8,146.8 (2 C), 149.6 (2C)) and two α, the carbon atom signal of beta-unsaturated carbonyl (δ 168.5 (2 C)) has further proved chemical compound Z
2In contain two coffee acyl structure fragments.In addition, δ 5.68 (1H, brs), 5.16 (1H, brs) and 4.27 (1H, brs) three even oxygen hydrogen and 2.16 (2H, m) and 2.03 (2H, m) four proton signals, and
13Two mesomethylene carbons (δ 39.1,37.7) among the C NMR, (δ 75.3 for the methine carbon of 3 company's oxygen, 70.1,67.0), a quaternary carbon (δ 75.9) and a carboxylic carbonyl carbon signal (δ 175.1), closely similar with the chemical shift of the quinic acid of reporting in the document.And chemical compound Z
2The C-3 and the C-4 of quinic acid structure fragment have the acidylate displacement, thereby can infer chemical compound Z
2Parent nucleus should be quinic acid, and have two coffee acyl substituent groups to be associated in 3 and 4 of quinic acid parent nucleus respectively.In the HMBC spectrogram, δ 5.68 (H-3), the carbon of 5.16 (H-4) and δ 168.5 (C-9 ') has long-range coherent signal, and therefore two coffee acyls are connected in 3 and 4 of quinic acid.As checkings such as HSQC, HMBC, NOESY, finally determine the caffeoyl guinic acid compounds Z in the Herba Taraxaci extract effective site through the two dimensional NMR spectrum
2Be 3, the 4-O-dicaffeoyl quinic acid.
2.6.3 4,5-O-dicaffeoyl quinic acid (Z
3):
1. mass spectrum: anion FABMS shows quasi-molecular ion peak m/z 515[M-1]
-, 353[M-H-162 (coffee acyl)]
-, 191[M-H-162 (coffee acyl)-162 (coffee acyl)]
-, 173[M-H-162-162-18 (H
2O)]
-
2. infrared: chemical compound Z
3IR spectrum and chemical compound Z
1Extremely similar, it is shown with hydroxyl (3445cm
-1), ester group (1694cm
-1), phenyl ring (1597,1524cm
-1) and trans double bond (978cm
-1).
3.
1H,
13CNMR and DEPT spectrum can be released chemical compound Z
3Molecular formula be C
25H
24O
12, chemical compound Z
3 1Also provided among the H NMR two groups of coffee acyl protons characteristic signal δ 6.99 (1H, brs), 6.96 (1H; brs), 6.88 (1H, d; J=8Hz), 6.85 (1H, d; J=8Hz), 6.71 (2H, m) and the trans olefinic proton signal δ 6.25 of two groups of AB types; (6.15 each 1H, d, J=16.0 Hz) and δ 7.56; 7.48 (each 1H, d, J=16.0Hz).
13C NMR and DEPT spectrum are providing ten undersaturated methine carbons (δ 115.1 (2CH) between δ 114-169,116.4 (2CH), 123.2 (2 CH), 147.5,147.7 (3CH)), (δ 127.5,127.6,146.7 (2 C) for six fragrant quaternary carbons, (149.6 2 C)) and two α, the carbon atom signal of beta-unsaturated carbonyl (δ 168.6,168.3) has further proved chemical compound Z
3In contain two coffee acyl structure fragments.In addition, 5.74 (1H, brs), 5.09 (1H, d, J=7.2) and 4.35 (1H, brs) three even oxygen hydrogen and 2.26 (2H, m), 2.10 (1H, m) and 2.26 (1H, m) four proton signals, and
13Two mesomethylene carbons (δ 38.5,39.8) among the C NMR, (δ 76.1 for the methine carbon of 3 company's oxygen, 69.8,69.2), a quaternary carbon (δ 76.2) and a carboxylic carbonyl carbon signal (δ 176.1), closely similar with the carbon atom chemical shift of the quinic acid of reporting in the document.And chemical compound Z
3The C-4 and the C-5 of quinic acid structure fragment have the acidylate displacement, thereby can infer chemical compound Z
3Parent nucleus should be quinic acid, and have two coffee acyl substituent groups to be associated in 4 and 5 of quinic acid parent nucleus respectively.
4.HMBC in the spectrogram, δ 5.09 (H-4), the carbon of 5.74 (H-5) and δ 168.3,168.6 has long-range coherent signal, and therefore two coffee acyls are connected in 4 and 5 of quinic acid.As checkings such as HSQC, HMBC, NOESY, finally determine the caffeoyl guinic acid compounds Z in the Herba Taraxaci extract effective site through the two dimensional NMR spectrum
3Be 4, the 5-O-dicaffeoyl quinic acid.
Embodiment 2: the preparation of the Herba Taraxaci extract spray powder raw material glue wafer for preparing by embodiment 1
Use 1 that prepare by embodiment, contain the pharmaceutical composition 10 in the claim 1～6,000mg, with raw material: starch: microcrystalline Cellulose is inserted in after granulate (1: 0.5: 0.5) in proportion in No. 1 Capsules.Adorn 100 altogether, the heavy 200mg of each capsule.
Embodiment 3: the preparation of the Herba Taraxaci extract spray powder feedstock composition capsule for preparing by embodiment 1
Use 1 that prepare by embodiment, contain the pharmaceutical composition 5 in the claim 1～6,000mg, with raw material: starch: microcrystalline Cellulose is inserted in after granulate (1: 1: 1) in proportion in No. 2 Capsuleses.Adorn 100 altogether, the heavy 150mg of each capsule.
Herba Taraxaci extract spray powder raw material has important biological, the present invention adopts micro-cytopathic-effect inhibition assay and dimethyl diaminophenazine chloride staining with this Herba Taraxaci extract spray powder raw material, the plaque method, chick embryo method, with the positive contrast medicine of ribavirin (virazole), from the maximal non-toxic concentration of Herba Taraxaci extract spray powder raw material, measure the medium effective concentration (EC that Herba Taraxaci extract spray powder raw material suppresses influenza virus FM-1 strain
50) and therapeutic index.Find that Herba Taraxaci extract spray powder raw material has an effect that suppresses influenza virus FM-1 more by force external.
Herba Taraxaci extract spray powder raw material of the present invention can combine with spoke material or carrier pharmaceutically commonly used, prepares medicine and pharmaceutical composition or health product with disease that prevention and treatment influenza virus cause.Above-mentioned various kinds of drug compositions or health product can adopt drug forms such as tablet, capsule, injection, aerosol, suppository, membrane, drop pill, externally-applied liniment, ointment.
Herba Taraxaci extract spray powder raw material of the present invention can also be united use with the medicine of influenza virus inhibitor that has now gone on the market or other treatment influenza associated conditions and crude drug thereof such as amantadine and rimantadine and/or neuraminidase inhibitor such as zanamivir and Oseltamivir etc., prepare and have the treatment active compositions of influenza associated conditions or compound preparation, can expect becomes treatment influenza disease medicine or health product.Above-mentioned various kinds of drug compositions or health product can adopt drug forms such as tablet, capsule, injection, aerosol, suppository, membrane, drop pill, comprise the conventional preparation of pharmaceutics general knowledge that employing has now been generally acknowledged and various slow release, controlled release form or the nanometer formulation that gets.
In order to understand essence of the present invention and the application prospect of Herba Taraxaci extract phenolic acid effective site of the present invention on drug development better, introduce the Herba Taraxaci extract effective site of preparing among the present invention respectively with pharmacology embodiment form below and adopt micro-cytopathic-effect inhibition assay and dimethyl diaminophenazine chloride staining, the plaque method, chick embryo method, with the positive contrast medicine of ribavirin (virazole), from the maximal non-toxic concentration of Herba Taraxaci extract spray powder raw material, measure the medium effective concentration (EC of Herba Taraxaci extract spray powder raw material resisiting influenza virus FM-1 strain
50), The pharmacological results such as therapeutic index illustrate its new purposes in pharmaceutical field.Pharmacology embodiment has provided the part activity data of representative Herba Taraxaci extract phenolic acid effective site.Mandatory declaration, pharmacology embodiment of the present invention and the pharmacodynamics model that is adopted are to be used to illustrate the present invention rather than limitation of the present invention.The simple modifications that essence according to the present invention is carried out the present invention, and other indications of the simple and easy expansion on the cause of disease basis that the embodiment of the invention contained all belong to the scope of protection of present invention.
Pharmacology embodiment 1:The resisiting influenza virus effect of the Herba Taraxaci extract spray powder raw material that trace cytopathy inhibition (CPE) method research embodiment 1 prepares
1.1 Herba Taraxaci extract spray powder raw cell toxicity test
Get one bottle of eugonic mdck cell, cultivate into cell monolayer.Discard nutritional solution, wash 2 times with PBS.
With the Herba Taraxaci extract spray powder raw material stock solution that has prepared, make continuous 10 times serial dilution with serum-free 1640 liquid respectively, dilute 6 concentration, and be labeled as stock solution (100mg/ml), 10mg/ml, 1mg/ml, 100 μ g/ml, 10 μ g/ml, 1 μ g/ml, 7 concentration of 0.1 μ g/ml.Then stock solution and 6 concentration liquids are added respectively in the mdck cell plate, and establish the normal cell contrast.Whole experiment repeats 3 times.96 orifice plates are put 37 ℃, 5%CO
2Cultivated 3 days in the incubator.Observation of cell pathological changes effect (CPE), outcome record is: " ++ ++ " be 100% pathological changes; " +++" be 75% pathological changes; " ++ " is 50% pathological changes; "+" is 25% pathological changes, "-" negative result.To preserve after the 96 orifice plate violet staining after observing end.Finally determine Herba Taraxaci extract spray powder raw material pair cell median toxic concentration (TD as calculated
50) be 3.22mg/mL, 90% poisoning concentration is 8.18mg/mL, maximal non-toxic concentration is 1000 μ g/ml.
1.2 micro-cytopathic-effect inhibition assay is measured the effect of Herba Taraxaci extract spray powder raw material extracorporeal antivirus effect
Get one bottle of eugonic monolayer mdck cell, add 1640 dilutions, concentration is 100TCID
50Virus liquid 100 μ l, 37 ℃, 5%CO
2Under adsorbed 2 hours.Add Herba Taraxaci extract spray powder raw material maximal non-toxic concentration (TD
0) medicinal liquid makes 6 concentration of continuous 2 times of dilutions, every concentration 3 holes, 37 ℃, 5%CO
2Under cultivated 3 days.Add ribavirin (virazole) maximal non-toxic concentration (TD
0) medicinal liquid makes 6 concentration of continuous 2 times of dilutions, 37 ℃, 5%CO
2Under cultivated 3 days.Set up experimental drug and positive drug, cell contrast and virus control group respectively.Microscopically observation of cell pathological changes after 3 days, record lesion degree (CPE).Calculate the antiviral effect of Herba Taraxaci extract spray powder raw material and ribavirin respectively by the Reed-Muench method.The medium effective concentration IC of the Herba Taraxaci extract spray powder raw material of measuring according to the CPE method
50=238 μ g/ml; Therapeutic index TI=TD
50/ IC
50=13.5; And the medium effective concentration 50.0 μ g/ml of the ribavirin of positive control; Therapeutic index TI=253.0.
Pharmacology embodiment 2:The resisiting influenza virus effect of the Herba Taraxaci extract spray powder raw material that dimethyl diaminophenazine chloride staining research embodiment 1 prepares
2.1 Herba Taraxaci extract spray powder raw cell toxicity test: with pharmacology embodiment 1.2.2 get one bottle of eugonic monolayer mdck cell, add 1640 dilutions, concentration is 100TCID
50Virus liquid 100 μ l, 37 ℃, 5%CO
2Under adsorbed 2 hours.Add Herba Taraxaci extract spray powder raw material maximal non-toxic concentration (TD0) medicinal liquid and make 6 concentration of continuous 2 times of dilutions, every concentration 3 holes, 37 ℃, 5%CO
2Under cultivated 3 days.
Add ribavirin (virazole) maximal non-toxic concentration (TD
0) medicinal liquid makes 6 concentration of continuous 2 times of dilutions, 37 ℃, 5%CO
2Under cultivated 3 days.Set up experimental drug and positive drug, cell contrast and virus control group respectively.Wash the back with PBS after 3 days and add dimethyl diaminophenazine chloride dye liquor, 37 ℃, 5%CO
2Hatched 1.5 hours.Abandon dyeing liquor, clean with PBS again, every then hole adds 90% ethanol, and room temperature was placed 2-3 hour, by microplate reader, was blank with the virus control hole, in 546nm place colorimetric, measured its absorbance A value.Log.
The medium effective concentration 206 μ g/ml of the Herba Taraxaci extract spray powder raw material that calculates according to the dimethyl diaminophenazine chloride staining; Therapeutic index TI=TD
50/ IC
50=15.6; And the medium effective concentration 33.9 μ g/ml of the ribavirin of positive control; Therapeutic index TI=287.0.Dimethyl diaminophenazine chloride dyeing result of calculation and CPE observed result meet substantially, and the result shows that the antiviral effect of ribavirin is better than Herba Taraxaci extract spray powder raw material.
Pharmacology embodiment 3:The resisiting influenza virus effect of the Herba Taraxaci extract spray powder raw material that plaque inhibition method (PFU) research embodiment 1 prepares
3.1 Herba Taraxaci extract spray powder raw cell toxicity test: with pharmacology embodiment 1.
3.2 plaque suppresses method
Get three bottles of eugonic monolayer mdck cells, clean, add the good 100TCID of dilution in advance with PBS
50FM-1 virus liquid, the every hole of 100 μ l/, 37 ℃, 5%CO
2Under adsorbed 1.5 hours.With Herba Taraxaci extract spray powder raw material from maximal non-toxic concentration (TD
0) beginning, remake 5 concentration of continuous 2 times of dilutions, add respectively in above-mentioned 24 orifice plates.37 ℃, 5%CO
2Under cultivated 40 hours.The culture plate cell adds formalin fixed, and the plaque counting is carried out in crystal violet solution dyeing.Compare with virus control, calculate 50% inhibition concentration IC
50Experiment repeats 3 times.Try to achieve mean P FU.Compare with virus control, calculate 50% inhibition concentration IC
50Be 300 μ g/ml, promptly Herba Taraxaci extract spray powder raw material can suppress the plaque formation of FM-1 strains of influenza viruses 50% when 300 μ g/ml.
Pharmacology embodiment 4: chick embryo method is measured the inhibitory action of the medicine of the Herba Taraxaci extract spray powder raw material that is prepared by embodiment 1 to influenza virus
4.1 influenza virus is to the median infective dose of Embryo Gallus domesticus: the dilution influenza virus of difference (IVA) is inoculated in respectively in the Embryo Gallus domesticus, collects allantoic fluid,, measure virus median infective dose (EID in Embryo Gallus domesticus by hemagglutination test
50), to understand the virulence situation of IVA in Embryo Gallus domesticus, adopt the Reed-Muench method to calculate the EID of IVA in Embryo Gallus domesticus
50Be 10
-3/ 0.1mL, in the antiviral experiment, the virus attack amount of employing is 100 EID
50
4.2 medicine is to the toxic action of Embryo Gallus domesticus: for understanding Herba Taraxaci extract spray powder raw material, ribavirin toxic action to Embryo Gallus domesticus, dosage in the confirmed test, to be subjected to reagent and each concentration of contrast medicine (100mg/mL, 10mg/mL, 1mg/mL, 100 μ g/mL) inoculate 5 Embryo Gallus domesticus, 0.25mL/ embryo respectively.Cultivate after 5 days for 35 ℃ and observe.Dead 2 of Herba Taraxaci extract spray powder raw material group original liquid concentration (100mg/mL), the concentration that Herba Taraxaci extract spray powder raw material 100mg/mL is described has certain toxicity to Embryo Gallus domesticus, chicken embryo death does not appear in other each concentration, illustrates that the Herba Taraxaci extract spray powder raw material of these concentration is less to Embryo Gallus domesticus free of toxic effects or toxicity; Ribavirin group chick embryo development is generally less, and dead 5 of original liquid concentration illustrates that the concentration of ribavirin 100mg/mL is bigger to Embryo Gallus domesticus toxicity, and chicken embryo death does not appear in other each concentration, illustrates that the ribavirin of these concentration is less to Embryo Gallus domesticus free of toxic effects or toxicity.Two kinds of medicines are 10mg/mL to the maximal non-toxic concentration of Embryo Gallus domesticus.
4.3 chick embryo method is measured the inhibitory action of medicine to influenza virus:
The dilution Herba Taraxaci extract spray powder of difference raw material is injected 0.25mL respectively in Embryo Gallus domesticus, and half an hour is after identical approach injection 100EID
50The viral 0.1mL of median infective dose was hatched 5 days.Collect allantoic fluid, survey its hemagglutinative titer titre.Can suppress the minimum dose that its blood clotting titre is injected more than 32 times, be its inhibition tire (MIC).Adopt the accurate probability inspection method of Fisher, relatively the infection rate of each treatment group.The result shows that Herba Taraxaci extract spray powder material concentration is that 10mg/ embryo dosage group has the obvious suppression effect to influenza virus.Ribavirin 10mg/ embryo, 1mg/ embryo dosage group all have obvious inhibitory action, and as can be seen, antivirus action is lower than ribavirin in the Herba Taraxaci extract spray powder raw material Embryo Gallus domesticus, its inhibition MIC=10mg/mL that tires.
Above antivirus test embodiment result illustrates that all Herba Taraxaci extract spray powder raw material has obvious effect on prevention and treatment influenza virus infectious disease.Be expected to be developed further into new Chinese medicine into prevention and treatment influenza disease.
Claims (10)
1. one kind has the Herba Taraxaci plant extract that suppresses the influenza virus function, it is characterized by by following method to prepare:
A) with the Herba Taraxaci plant of solvent extraction through pulverizing or being cut into segment, concentration extraction reclaims solvent;
B) concentrated solution that step a is obtained is through column chromatography, and the alcoholic solvent eluting is used in washing then;
C) eluent that step b is obtained is evaporated to does not have the alcohol flavor, drying, pulverize product.
2. according to the Herba Taraxaci plant extract of claim 1, wherein the solvent among the step a is pure water mixed solvent, and wherein alcohol is methanol, ethanol, ethylene glycol or their mixture;
Column chromatography used medium among the step b is silica gel, reverse phase silica gel, macroporous resin, ion exchange resin, aluminium oxide or polydextran gel (Sephadex) class; Alcoholic solvent among the step b is meant ethanol water.
3. according to the Herba Taraxaci plant extract of claim 2, wherein the solvent among the step a is the ethanol water of 30-75%, extracts 1-5 time, doubly measures with 3-10 at every turn, extracts 0.5-3 hour at every turn; Column chromatography used medium among the step b is a macroporous resin; Alcoholic solvent among the step b is the ethanol of 30-80%.
4. according to the Herba Taraxaci plant extract of one of claim 1-3, the content that it is characterized by phenolic acid compound in the extract 20% and more than.
5. according to the Herba Taraxaci plant extract of one of claim 1-4, it is characterized by in this extract caffeic acid content 0.3% and more than; Luteolin 7-O-β-D-glucoside content 0.5% and more than.
6. according to the Herba Taraxaci plant extract of one of claim 1-4, it is characterized by in this extract except that containing caffeic acid, ferulic acid and chlorogenic acid, also contain two caffeoyl guinic acid class compound of phenolic acid active substances of three structural similarities, they are: 3, and the 5-O-dicaffeoyl quinic acid; 3, the 4-O-dicaffeoyl quinic acid; 4, the 5-O-dicaffeoyl quinic acid.
7. the Herba Taraxaci plant extract according to one of claim 1-6 has the pharmacological activity that suppresses influenza virus.
8. be used to prepare the influenza that prevention or treatment viral infection cause and the purposes of associated conditions medicine thereof according to the Herba Taraxaci plant extract of one of claim 1-6.
9. preparation-obtained a kind of medicine or the pharmaceutical composition with resisiting influenza virus function of purposes according to Claim 8 is characterized by Herba Taraxaci plant extract and pharmaceutically suitable carrier of containing one of claim 1-6 for the treatment of effective dose.
10. according to the medicine or the pharmaceutical composition of claim 9, it can be tablet, granule, capsule, oral liquid, injection, drop pill, transdermal patch or aerosol, can also adopt the known controlled release of modern pharmaceutical circle or slow release formulation or nanometer formulation.
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| CNA2006100366742A CN101112409A (en) | 2006-07-26 | 2006-07-26 | Preparation of phenolic acid valid target in dandelion and use thereof for inhibiting influenza virus |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101982184A (en) * | 2010-10-27 | 2011-03-02 | 安徽济人药业有限公司 | Preparation method of taraxacum extract |
| CN106265945A (en) * | 2016-09-21 | 2017-01-04 | 西安乐道生物科技有限公司 | A kind of SHUANGHUANLIAN effervescent granule for animals and preparation method thereof |
| CN107998166A (en) * | 2017-12-26 | 2018-05-08 | 湖南知达医药科技有限公司 | The preparation method of dandelion extract |
| CN110251499A (en) * | 2019-06-10 | 2019-09-20 | 陕西理工大学 | Dandelion extract and its purposes in preparation prevention and treatment woman vagina disease medicament |
| CN110320305A (en) * | 2019-08-14 | 2019-10-11 | 广西壮族自治区药用植物园 | Detection method while dandelion a variety of active ingredients |
| CN111514213A (en) * | 2020-04-01 | 2020-08-11 | 清华德人西安幸福制药有限公司 | Antiviral traditional Chinese medicine composition for respiratory system |
| CN113476492A (en) * | 2021-08-23 | 2021-10-08 | 盛春华 | Dandelion seed extract and application thereof |
| US11235015B2 (en) | 2017-05-16 | 2022-02-01 | Hongzhang Jia | Antiviral Traditional Chinese Medicine composition and preparation method and use i'hereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101982184A (en) * | 2010-10-27 | 2011-03-02 | 安徽济人药业有限公司 | Preparation method of taraxacum extract |
| CN106265945A (en) * | 2016-09-21 | 2017-01-04 | 西安乐道生物科技有限公司 | A kind of SHUANGHUANLIAN effervescent granule for animals and preparation method thereof |
| US11235015B2 (en) | 2017-05-16 | 2022-02-01 | Hongzhang Jia | Antiviral Traditional Chinese Medicine composition and preparation method and use i'hereof |
| JP7008368B2 (en) | 2017-05-16 | 2022-02-10 | ホンチャン ジア | Antiviral Chinese herbal medicine composition and its preparation method and use |
| CN107998166A (en) * | 2017-12-26 | 2018-05-08 | 湖南知达医药科技有限公司 | The preparation method of dandelion extract |
| CN110251499A (en) * | 2019-06-10 | 2019-09-20 | 陕西理工大学 | Dandelion extract and its purposes in preparation prevention and treatment woman vagina disease medicament |
| CN110251499B (en) * | 2019-06-10 | 2022-11-01 | 陕西理工大学 | Dandelion extract and application thereof in preparing medicine for preventing and treating woman vaginal diseases |
| CN110320305A (en) * | 2019-08-14 | 2019-10-11 | 广西壮族自治区药用植物园 | Detection method while dandelion a variety of active ingredients |
| CN110320305B (en) * | 2019-08-14 | 2022-04-15 | 广西壮族自治区药用植物园 | Method for simultaneously detecting multiple active ingredients of dandelion |
| CN111514213A (en) * | 2020-04-01 | 2020-08-11 | 清华德人西安幸福制药有限公司 | Antiviral traditional Chinese medicine composition for respiratory system |
| CN113476492A (en) * | 2021-08-23 | 2021-10-08 | 盛春华 | Dandelion seed extract and application thereof |
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